Analysis of antimicrobial resistance patterns and genetic mutations in Helicobacter pylori from West Bengal, India depicting escalating clarithromycin and high levofloxacin resistance

Abstract

Background

The growing resistance of Helicobacter pylori (H. pylori) to antibiotics poses a significant global health challenge, particularly in developing regions where infection rates are high, and routine antibiotic susceptibility surveillance is limited. This study aimed to evaluate the antimicrobial resistance patterns and associated genetic mutations in H. pylori strains isolated from patients in West Bengal (WB), India.

Methods

A total of 88 H. pylori strains were isolated from gastric biopsy samples collected between 2018 and 2020 from patients diagnosed with gastritis, duodenal ulcer, or gastric cancer. Antimicrobial susceptibility was determined using the agar dilution method against six antibiotics: metronidazole, tetracycline, clarithromycin, furazolidone, levofloxacin, and amoxicillin. Resistance-associated mutations in gyrA (levofloxacin), frxA and rdxA (metronidazole), and pbp1A (amoxicillin) were characterized via Sanger sequencing. Mutations in the 23SrRNA gene, implicated in clarithromycin resistance, were identified through allele-specific PCR, validated by Sanger sequencing and transformation assays. Additionally, BsaI and BbsI restriction enzyme digestion was used to confirm specific point mutations in 23SrRNA gene of clarithromycin-resistant isolates.

Results

Of 210 biopsies analyzed, 79 (37.6%) were H. pylori-positive, yielding 88 distinct strains—some patients harbored multiple genetically diverse isolates, identified by differences in cagA/vacA genotypes and antibiotic resistance profiles. Although, no association was found between virulence gene profile and resistance pattern. Resistance rates were highest for levofloxacin (69.3%), followed by metronidazole (61.4%) and clarithromycin (19.3%). Nearly all clarithromycin-resistant isolates carried the A2143G mutation in the 23SrRNA gene, capable of getting naturally transmitted into the next generations also. Levofloxacin-resistant strains harbored mutations in GyrA at codons 63, 87, 88, 91, 130, and 150, and a novel Gly85Ala substitution was identified. Metronidazole resistance correlated more strongly with rdxA mutations than with frxA. No resistance was detected against furazolidone and tetracycline, while amoxicillin resistance was rare (1.1%). Multidrug resistance was observed in 10.2% of isolates, with 32.9% displaying dual resistance to metronidazole + levofloxacin, and 6.8% to clarithromycin + levofloxacin.

Conclusions

High clarithromycin, substantial metronidazole and levofloxacin resistance in H. pylori strains from WB underscore the urgent need to revise empirical treatment strategies. These findings advocate for localized, resistance-guided therapy protocols and reinforce the importance of continuous antimicrobial surveillance to optimize treatment outcomes and mitigate resistance development.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13099-025-00744-x.

Background

Helicobacter pylori (H. pylori) is a Gram-negative, micro-aerophilic bacterium that colonizes the human stomach and is primarily transmitted through contaminated food and water infecting approximately 50% of the global population [1, 2]. This bacterium is implicated in a wide spectrum of gastroduodenal diseases including mild gastritis, peptic ulcer (PU) or duodenal ulcer (DU), gastric cancer (GC), mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori infection is notably higher in developing countries such as India with reported infection rates ranging from 49.9% to 83.3% [3]. Among the most studied H. pylori virulence factors are the cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA), both of which are strongly linked to severe clinical outcomes. The cagA gene was the first H. pylori virulence gene to be characterized and is located at the terminal region of the horizontally acquired cag pathogenicity island PAI (cag PAI). This protein is known to interfere with host cell signaling pathways, including β-catenin and ERK (Extracellular signal- regulated kinase pathway), thereby promoting inflammation and cellular dysregulation. In contrast, VacA is a uniquely multifunctional toxin that induces the formation of large cytoplasmic vacuoles in host cells, modulates immune responses, disrupts cellular signaling, and contributes to apoptosis [4–6]. While surveillance of these virulence genes can provide valuable insights into the epidemiology of H. pylori infections, current evidence suggests that eradication therapy is a more cost-effective approach than widespread molecular screening [7, 8]. Therefore, individuals presenting with gastroduodenal disorders in conjunction with H. pylori infection should be prioritized for eradication treatment.

A variety of antibiotic-combination therapies, including metronidazole, amoxicillin, clarithromycin, levofloxacin, furazolidone, tetracycline, and rifabutin are formulated as no single antibiotic has demonstrated > 80% eradication efficacy against H. pylori in clinical settings [9–11].

Treatment guidelines in India, aligned with MaastrichtVI/Florence Report (2022), primarily recommend PAC-Triple Therapy regimen which includes a Proton Pump Inhibitor (PPI), amoxicillin, and clarithromycin. In regions with high clarithromycin resistance, bismuth - quadruple therapy (PPI + Bismuth + Metronidazole + Tetracycline) is the preferred alternative [12]. Clarithromycin has long served as a cornerstone of H. pylori eradication; however, the growing prevalence of clarithromycin-resistant strains has significantly compromised its efficacy. In such cases, particularly where bismuth-based regimens also prove ineffective, a levofloxacin-based triple therapy (PPI + levofloxacin + amoxicillin) is recommended as a second-line approach [13].

Globally, there is a substantial variation of H. pylori antibiotic resistance profiles. Nevertheless, a consistent trend over the past two decades reveals a steady rise in resistance, accompanied by a decline in treatment success rates to approximately 65–80% [12, 14, 15]. Patients are often prescribed empirical first-line therapies without prior antibiotic susceptibility testing. The widespread, unregulated use of these antibiotics for unrelated infections, such as respiratory, urinary, genital, or parasitic diseases, further contributes to the emergence of resistant H. pylori strains and incomplete eradication [15, 16].

H. pylori is exceptionally resilient to any change in micro-environment, and mostly gains antimicrobial resistance by chromosomal genetic mutations rather than plasmid-mediated mechanisms. These mutations typically interfere with the drug's activity by altering its target site or inhibiting its activation, thereby facilitating persistent infection despite treatment efforts [14, 16].

Mukhopadhyay et al., [1] demonstrated that certain virulence-associated genes of the Kolkata H. pylori population are distinctly different from that of other global regions, likely due to natural selection and founder effects. These evolutionary forces were also reflected in the antibiotic resistance profiles, with significant differences observed between H. pylori isolates from Kolkata and those from Japan or Western countries [1]. Notably, since 2005, no comprehensive antibiotic resistance profiling has been conducted for the Kolkata population. The 2005 study reported no resistance to amoxicillin or clarithromycin among local strains, although high resistance to metronidazole (85%) and moderate resistance to tetracycline (7.5%) were observed [17]. However, more recent data suggest an emerging trend of antibiotic resistance, with clarithromycin resistance in Kolkata reported at 8.04% [18].

Due to irregular antibiotic susceptibility surveillance across India and absence of a coordinated national program, therapeutic recommendations often rely on insufficient or geographically unrelated locations’ data. The rising resistance to commonly used antibiotics, combined with significant regional variations in resistance patterns and a critical lack of current data from West Bengal (WB), necessitates a localized investigation. This study therefore aims to assess the prevalence of antimicrobial resistance among H. pylori strains from West Bengal, including the Kolkata region, and to elucidate the genetic mechanisms underlying these resistance phenotypes.

Methods

Patients details and procurement of endoscopy tissue samples

Patients seeking diagnosis at Manipal Hospitals Broadway, Kolkata, with gastroduodenal diseases like DU, PU, functional dyspepsia, and GC, during years 2018–2020 were included in this study.

Exclusion criteria included patients with prior H. pylori treatment; with co-morbid conditions such as HIV/AIDS, tuberculosis, or malignancies under chemotherapy; and individuals unwilling to provide informed consent. Elimination criteria included inadequate or poor-quality biopsy samples, contaminated, and incomplete clinical data or loss to follow-up.

They were from both sex groups; aged between 14–72 yrs. Last date of sample collection was 18^th March, 2020, before COVID-19 cases were reported from this study population. With informed consent, two tissues each from antrum and corpus were collected. Rapid urease test (RUT) was done with one tissue, another part stored for culturing, in ice cold transport media [Glycerol(15%) + Brucella broth] and stored at – 80 ℃, within 2–3 hrs of collection. This study has been approved by ICMR-NICED ethical committee. Identity of patients were not disclosed during experimental procedures.

Processing of biopsy samples and culturing of H. pylori

The thawed tissue samples were vortexed for 15 mins following which 100–150 µl of transport media was plated onto Brain Heart Infusion Agar (BHIA) + charcoal (0.2%) agar plates and incubated in microaerobic condition for 3–7 days. The water droplet like transparent H. pylori colonies were identified and further confirmed via urease, catalase and oxidase reactions. Each time H. pylori reference strain 26695 was used as an experimental positive control. From each biopsy specimen, 10 single and one pooled colonies were picked, and sub-cultured on alternate days onto fresh BHIA plates. One loopful of bacterial culture was then stored at – 80 ℃ in BHI broth + glycerol (20%), as mentioned earlier [2].

Extraction of H. pylori genomic DNA

Loopful bacterial culture was harvested and DNA was isolated using CTAB (Cetyltrimethylammonium bromide)—method with phenol–chloroform as mentioned previously [19] and stored at – 20 ℃.

Genotypic characterization of H. pylori isolates

PCR amplification of ureB using gene-specific primers was performed for further confirmation of H. pylori infection. Simultaneously, PCR amplification of cagA and vacA (s and m alleles) were carried out using gene-specific primers (Table 1) to characterize the genotypes of the infecting strains. This also provided insight into the possibility of multiple strain infections within a single host [2, 20].

Table 1.

List of primers

Gene/gene region	Primer	Sequence (5′−3′)	Tm (℃)	Reference
cagA	cag5c F	GTT GAT AAC GCT GTC GCT TCA	55	[2]
	cag3c R	GGG TTG TAT GAT ATT TTC CAT AA	55
vacA m1/m2	vag F	CAA TCT GTC CAA TCA AGC GAG	55
	vag R	GCG TCA AAA TAA TTC CAA GG	55
vacA s1/s2	va1 F	ATG GAA ATA CAA CAA ACA CAC	55
	va1 R	CTG CTT GAA TGC GCC AAA C	55
cag PAI empty site	Lunil 1	ACA TTT TGG CTA AAT AAA CGC TG	55
	R5280	CCA ACG TGC GTA AAA GGG AAT TAG	55
gyrA	gyrAF	GTA GAA GTG GGG ATT GAT TCT TC	55	This Study
	gyrAR	GCG TCT ATG ATT TCA TCC ATC CTG	55
pbp1a	AMX RES F	GGC TAT ACC ATA AAG CTC ACG AT	55
	AMX RES R	GAG ATA GCA AAG CTC CCT AAC AC	55
	AMX RESII F	TTG AGC CAT TCG TTA AAT CTA GCC	55
	AMX RESII R	TTA GAA CAA CAA GCG TTC CTC G	55
frxA	frxA-F11	GCT TTA CAG CAC CAA CGA TTT G	55	[23]
	frxA-R16	TAA ATA ACT TCT GTC TTC CAG CGG	55
rdxA	rdxAseq F	CTC GCT TCT GCC ACC CTC TT	60	This study
	rdxAseq R	CGC CAT TCT TGC AAG ATG TTT GA	60
23Sr RNA	23SrRNA F	GGC TCT TTG AGT CCT TTT AGG ACA A	60	[18]
	23SrRNA R	CTC CAT AAG AGC CAA AGC CCT TAC T	60
	2143F	GTA AAC GGC GGC CGT AAC TAT	60	[17]
	2143AR	GTA AAG GTC CAC GGG GTC GT	60
	2143GR	GTA AAG GTC CAC GGG GTC GC	60
ureB	ureB F	CGT CCG GCA ATA GCT GCC ATA GT	50
	ureB R	GTA GGT CCT GCT ACT GAA GCC TTA	50

The primers used in this study for PCR amplification of various genes

Additionally, strains which tested negative for cagA by PCR were further screened using cag-empty site PCR with specific primers (Luni1 and cagAR5280) to confirm the absence of the entire cag pathogenicity island (cagPAI). The primers as mentioned in Table 1. PCR was done in 20µl reaction volume, with respective conditions for each gene [2, 18] in a Master Cycler apparatus (Eppendorf, Hamburg, Germany). The amplified products were analyzed by 2% agarose gel electrophoresis against molecular markers (NEB) and imaging with analysis was done using Bio-Rad GelDoc Go.

Antimicrobial susceptibility assay

All the antibiotics used in this study (Supplementary file 1) were purchased from Sigma Chemicals, USA. Minimum inhibitory concentrations (MICs) for all antibiotics were determined by Agar-dilution method. All the dosages of antibiotics based upon which this study was conducted were in accordance with previous study [17] and EUCAST guidelines [20]. Isolates were considered to be resistant when the MIC was > 8 µg/ml for metronidazole, > 1 µg/ml for tetracycline, > 0.5 µg/ml for clarithromycin, > 1 µg/mlfor levofloxacin and > 0.125 µg/ml for amoxicillin as per EUCAST guidelines [21]. Whereas for furazolidone isolates were considered to be resistant when the MIC was > 2 µg/ml [17]. BHIA plates containing different concentrations of each antibiotic were prepared by adding the appropriate amount of the drug from their respective stock solutions. Frozen H. pylori cultures were streaked on a antibiotic—free BHIA plate and incubated under microaerobic conditions at 37 ℃ for 2 days. Then bacterial growth was spread on fresh antibiotic—free BHIA plate and incubated for 1 day. The resulting young exponentially growing cells were suspended in Phosphate Buffered Saline (PBS); serial 10- fold dilutions of these suspensions were prepared and 10 µl of each dilution was spotted on freshly prepared antibiotic supplemented as well as on drug free plate used as a control. All plates were incubated for 72 hrs. A strain was considered to be susceptible to concentrations of drugs that decreased its efficiency of colony formation at least 10 folds for metronidazole [24] and 1000-folds for rest of the tested antibiotics, as compared with the control plate [18].

Restriction enzyme digestion of 23SrRNA gene

All the clarithromycin resistant strains and representative sensitive strains were PCR-amplified using primers 23SrRNA F/R generating the 617 bp product as done previously, [19] with few modifications. This purified PCR product was subjected to restriction enzyme digestion as per manufacturer’s protocol with BsaI-HFv2 and BbsI (NEB). Digested products were electrophoresed in 2.5–3% agarose gel and analysed as mentioned before.

Nucleotide sequencing of H. pylori antibiotic resistance determining genes for detection of point mutation

All the PCRs (50 µl reaction volume) were performed using TaKaRa Taq polymerase with respective primers as mentioned in Table 1.

gyrA F/R were used for amplifying the 554 bp Quinolone Resistance Determining Region (QRDR) segment of gyrA (GenBank accession no. CP003904.1; locus tag: C694_03615) to detect point mutations responsible for levofloxacin resistance [22].

Similarly, for detection of point mutations related to amoxicillin resistance the pbp1a gene (GenBank accession no. AE000511.1: complement 630840..632819; locus tag: HP_0597) was PCR amplified in two fragments with primers AMX RES F/R and AMX RESII F/R.

rdxA (oxygen-insensitive NAD(P)H nitroreductase gene GenBank accession no. AE000511.1:complement 1013553.1014185; locus tag: HP_0954) and frxA, were PCR amplified for metronidazole-resistant strains.

Clarithromycin resistance related mutations in 23SrRNA gene were detected with PCR amplified 617bp product, using primer pair 23SrRNA F/R.

Amplified PCR product were then purified, using purification kit (GeneJET PCR purification Kit, ThermoFisher Scientific) and sequenced as mentioned earlier [1].

The sequenced data was assembled for each gene from respective forward and reverse sequence reads using FinchTV (Geospiza, Inc.) and were compared against reference strain 26695 (GenBank accession: CP003904.1), based on which all the primers were designed. DNA sequences were aligned with strain 26695 using the ClustalW Multiple Sequence Alignment tool to identify single nucleotide mutations. Corresponding protein sequences were generated using the ExPASy Translate Tool and the derived protein sequences were further analyzed using NCBI protein BLAST (BLASTp) to confirm protein identity and to access any amino acid changes resulting from non-synonymous mutations.

Statistical analysis

The correlation between the respective mutation and its association in development of antibiotic resistance were statistically justified using Cohen’s Kappa co-efficient analysis through the GraphPad (quantify interrater agreement with Kappa) online tool.

Results

Prevalence of H. pylori in WB

In this study, out of 210 biopsy samples collected (161 from male and 49 from female patients), H. pylori was successfully cultured from 79 (37.6%) samples. Among these, 69 samples were positive for both RUT and culture, while the remaining 10 samples were culture positive but RUT negative.

The observed discordance between RUT and culture results may be attributed to the patchy distribution of H. pylori within the gastric mucosa. It is likely that the biopsy sample used for RUT contained an insufficient bacterial load to produce a detectable urease reaction, whereas the parallel specimen used for culture harbored a higher concentration of viable organisms, resulting in successful isolation. Another possible explanation involves the frequent use of over-the-counter proton pump inhibitors (PPIs) by patients experiencing gastroduodenal symptoms before clinical consultation. PPI use is known to reduce the sensitivity of urease-based diagnostics [13]. Consequently, biopsy specimens obtained during endoscopy from such patients may yield false-negative RUT results, even though cultures from the same patients may show positivity.

Assessment of multiple strain infections within individual hosts was conducted using a combination of virulence gene profiling (cagA presence/absence and vacA allelic subtypes: m1/m2, s1/s2) and antimicrobial susceptibility testing. Out of 210, 71 cases were found to have H. pylori infection with single strain. One of the patients, exhibited infection with 3 distinct strains by virtue of having varying antibiotic susceptibility profile and genetic background from its antrum and corpus biopsy tissues. Dual infections were identified in 7 cases yielding 14 strains. Among these 7, 4 cases showed clear evidence of co-infection with genetically distinct H. pylori strains, as indicated by differences in both virulence gene profiles and antibiotic susceptibility patterns. While the remaining 3 cases exhibited heterogeneity solely in their antibiotic resistance profiles, suggestive of mixed-strain colonization. In total, 88 (71 + 3 + 14 = 88) H. pylori strains were isolated from the 79 positive biopsy samples and included in this study. Of 88 strains, 8 strains (9%) were susceptible to all the antibiotics tested. A comprehensive summary of the findings is provided in Table 2 and illustrated in Figure 1.

Table 2.

Showing the numerical distributional of Sensitive and Resistant strains

Sensitive strains	Single antibiotic resistant strains (n = 35)	Dual antibiotic resistant strains (n = 36)	Triple antibiotic resistant strains (n = 9)
8	15 (MET) 17 (LEV) 3 (CLA)	29 (MET + LVX) 6 (CLA + LVX) 1 (MET + CLA)	8 (MET + CLA + LVX) 1(MET + LVX + AMX)

The distribution of H. pylori strains (n = 88) isolated in West Bengal during the study period from 2018 to 2020, categorized by their antibiotic resistance profiles. Strains were classified as single-drug resistant, dual-drug resistant, or multidrug-resistant (MDR), based on their susceptibility patterns. Notably, eight isolates were found to be sensitive to all antibiotics tested. The antibiotics included in this analysis were Metronidazole (MET), Levofloxacin (LVX), Clarithromycin (CLA), and Amoxicillin (AMX)

Fig. 1.

Pattern of antibiotic susceptibility and resistance of H. pylori in West Bengal in 2018–2020: Distribution of MICs among the clinical Helicobacter pylori strains from WB isolated during 2018–2020 to Metronidazole (A), Tetracycline (B), Clarithromycin (C), Furazolidone (D), Levofloxacin (E), and Amoxicillin (F)

Escalating rate of clarithromycin resistance in WB-isolates:

Antimicrobial sensitivity of clarithromycin

Out of the 88 H. pylori isolates tested, 17 (19.3%) exhibited resistance to clarithromycin at a concentration of 0.5 µg/ml. The remaining 71 isolates (80.7%) were classified as sensitive, with MICs ranging from 0.125 to ≤ 0.5 µg/ml (Fig. 1).

PCR-based detection of A2143G mutation of 23SrRNA, conferring resistance

To further characterize the molecular basis of clarithromycin resistance, we screened all 88 isolates for the presence of the A2143G point mutation in the 23SrRNA gene. A previously standardized Mismatch Amplification Mutation Assay PCR (MAMA-PCR), developed in our laboratory [18], was employed for this purpose due to its rapidity and sensitivity.

Of the 17 phenotypically resistant isolates, 15 produced a positive amplification signal consistent with the presence of the A2143G mutation (Fig 2). However, two clarithromycin-resistant isolates did not yield a positive result in the MAMA-PCR assay.

Fig. 2.

Detection of clarithromycin resistance specific point mutation via single step PCR: Gel Image showing specific detection of 23SrRNA gene A2143G mutation, via MAMA-PCR with primers 2143 F, 2143AR and 2143GR. Gel A: Sensitive allele specific PCR & Gel B: Resistant allele specific PCR. Lane 1: 100 bp Marker, Lane 2,3,4: clarithromycin sensitive strains, Lane 5,6,7,8,9,10,11: clarithromycin resistant strains possessing A2143G mutation and Lane 12: clarithromycin resistant strains having A2142G mutation

PCR–RFLP-based detection of 23SrRNA mutation in clarithromycin resistant H. pylori

To further validate the PCR-based findings, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis was performed using two restriction enzymes, BsaI and BbsI. Amplified 23SrRNA gene products (using 23SrRNA F/R primers) were digested with BsaI to detect the A2143G mutation (Fig. 3). In contrast, digestion with BbsI distinguished A2143G from the less common A2142G mutation, as the latter introduces a unique BbsI restriction site absent in the former (Fig. 4).

Fig. 3.

Detection of clarithromycin resistant specific A2143G mutation via PCR–RFLP: mutation of Gel Image showing specific detection of 23SrRNA gene A2143G mutation, via restriction enzyme digestion of 617 bp sized PCR amplified product by BsaI enzyme. Lane 1: 100 bp Marker, Lane 2: clarithromycin sensitive strain, Lane 3: clarithromycin resistant strain possessing only A2142G mutation and Lane: 4, 5, 6: clarithromycin resistant strains all having A2143G mutation

Fig. 4.

Detection of clarithromycin resistant specific A2142G mutation via PCR–RFLP: Gel Image showing specific detection of 23SrRNA gene A2142G mutation, via restriction enzyme digestion of 617 bp sized PCR amplified product by BbsI enzyme. Lane 1: 100 bp Marker, Lane 2: clarithromycin sensitive strain, Lane 3: strain having mixed infection with clarithromycin resistance A2142G mutation and sensitive population simultaneously, Lane 4: clarithromycin resistant strain possessing only A2142G mutation and Lane 5, 6: clarithromycin resistant strains all having A2143G mutation. The banding pattern for the A2142G mutation carrying strains are highlighted with dotted white lines

Sequencing confirms predominance of A2143G mutation among resistant isolates

To corroborate the genotypic data, sequencing of the peptidyl transferase region of the 23SrRNA gene was performed on a subset of isolates (seven clarithromycin-resistant and four clarithromycin-sensitive strains). Among the seven resistant strains that were sequenced, five strains harbored the A2143G mutation positive by MAMA-PCR, while the remaining two strains that were negative by MAMA-PCR were found to carry the A2142G mutation (Table 3). These findings confirm that the A2143G mutation is the predominant resistance mechanism in this population. In addition to the point mutations at positions 2143 and 2142, the other mutations noted in both the resistant and sensitive strains included A2182C, A1821G, G1826A, and T1830C. Among the sequenced clarithromycin resistant strains, three had all four mutations and two strains had three of them except A2182C. The rest of the two resistant strains carried only the A2143G mutation. However, any predictive association of these 4 mutations with resistance development were nullified with their presence among the four sequenced sensitive strains. Specifically, two sensitive strains had concurrent presence of A2182C, A1821G, G1826A mutations, and one strain with only T1830C, while the other had all the four A2182C, A1821G, G1826A, and T1830C mutations.

Table 3.

The mutational changes responsible for antibiotic resistance development

Antibiotic	Resistance associated gene	Mutations (nucleotide or amino acid)	No. of resistant strains sequenced
Clarithromycin	23Sr RNA	Nucleotide: A2143G; A2142G	5(A2143G) 2(A2142G)
Levofloxacin	gyrA (QRDR region)	Amino acid: S63P; G85A; N87K; N87Y, D91N; D91G; R130K; V150A	27
Amoxicillin	pbp1A	Amino acid: E406A, D535N, S543H, T556S	1

The single nucleotide changes associated with clarithromycin resistance, and the amino acid changes found in levofloxacin, amoxicillin, and metronidazole resistant H. pylori isolates from this study

Transformation study confirms A2143G mutation in 23SrRNA gene as the determinant of clarithromycin resistance

To functionally validate the role of the A2143G mutation in conferring clarithromycin resistance, natural transformation experiments were conducted. The PCR-amplified fragment (using primers 23SrRNA F/R) of the 23SrRNA gene from a clarithromycin-resistant isolate harboring the A2143G mutation was used to transform the clarithromycin-sensitive reference strain H. pylori 26695. Following established protocols [23], transformants were selected on plates containing 1 µg/ml clarithromycin. Sequencing of the 23SrRNA gene from the resulting transformants (data not shown) confirmed the presence of the A2143G mutation, identical to that in the donor strain.

Rising trend of metronidazole resistance

Resistance pattern of metronidazole

Among the 88 H. pylori isolates tested, 54 strains (61.4%) exhibited resistance to metronidazole, highlighting a concerning trend of rising resistance in the region. Specifically, 22 strains (25%) demonstrated MIC values at 8 µg/ml (classified as 8R, ≤ 16S), 29 strains (33%) at 16 µg/ml (16R, ≤ 32S), and 3 strains (3.4%) at 32 µg/ml (32R, ≤ 64S), indicating varying degrees of high-level resistance. In contrast, 16 isolates (18.2%) and 18 isolates (20.5%) were classified as sensitive, exhibiting MICs at 2R ≤ 4S µg/ml and 4R ≤ 8S µg/ml, respectively (Fig. 1).

rdxA gene status plays a predominant role in metronidazole resistance than frxA

Metronidazole resistance in H. pylori is primarily mediated by mutations in genes encoding oxygen-insensitive nitroreductases, particularly rdxA (HP0954) and frxA (HP0642), which are responsible for activating the prodrug into its bactericidal and mutagenic forms [24]. Sequencing of selected resistant isolates revealed that the functional status of the rdxA gene plays a more critical role in resistance development compared to frxA. Two highly resistant strains, A140C(1) and A92C(1), with MICs of 32R, ≤ 64S, exhibited significant alterations in rdxA: one had a premature stop codon, and the other displayed point mutations. Notably, the frxA gene remained unaltered in both cases, suggesting its limited role in conferring resistance.

Extremely high resistance to the quadruple therapy antibiotic levofloxacin

Resistance pattern of levofloxacin

Levofloxacin resistance was alarmingly high among the H. pylori isolates analyzed, with 61 out of 88 strains (69.3%) demonstrating resistance. The remaining 27 strains (30.7%) were sensitive, with MICs ranging between 0.125 µg/ml and ≤ 1 µg/ml (Fig 1). Among the resistant group: 23 strains (26.1%) exhibited MICs at 1R, ≤ 4S µg/ml; 21 strains (23.9%) at 4R, ≤ 8S µg/ml; 12 strains (13.6%) at 8R, ≤ 16S µg/ml; 4 strains had MICs at 16R, ≤ 32S µg/ml, and 1 strain showed a very high MIC of 32R, ≤ 64S µg/ml. These findings reflect a significant and rising trend of resistance to levofloxacin.

QRDR mutations in gyrA gene correlate with levofloxacin resistance

Due to the observed high levofloxacin resistance, the genetic basis in these recent isolates was investigated. The QRDR region of the gyrA gene in 27 randomly selected resistant isolates was sequenced, each strain with varying MIC values, using 26695 strain as the reference. The novel mutation at position G85A, instead of the previously reported G85C mutation was identified from this analysis [25]. The mutation V150A, previously reported in sensitive strain [26], was found in one of our resistant strains. However, its role in conferring resistance needs further investigation. Among the sequenced strains, eight exhibited single substitution at the 91^st amino acid (aa) (D91N/G); twelve strains with 87^thaa mutated (N87K); four had simultaneous mutations at 91^st (D91N/G) and another (G85A or R130K or V150A); and single strain had double mutations at 63^rdaa (S63P) and 87^thaa (N87K). Two strains had mutations at both the 87^th (N87K/Y) and 91^staa (D91N/G). The mutations responsible for levofloxacin resistance development are shown in Table 3.

Emergence of amoxicillin—resistant H. pylori isolate

Susceptibility profile of amoxicillin

Of the 88 H. pylori isolates examined, 87 (98.9%) were highly sensitive to amoxicillin, while a single isolate exhibited resistance. The resistant strain demonstrated an MIC beginning at 0.125 µg/ml and complete inhibition at ≤ 0.25 µg/ml. Although this MIC falls within a low range, it exceeded the defined breakpoint for susceptibility, thereby qualifying as resistant. Among the sensitive strains: 61 isolates (69.3%) displayed MICs between 0.03125 µg/ml and ≤ 0.0625 µg/ml, 26 isolates (29.6%) showed MICs ranging from 0.0625 µg/ml to ≤ 0.125 µg/ml.

Sequencing of pbp1a gene associated with amoxicillin resistance

To elucidate the molecular basis underlying amoxicillin resistance, the  single resistant isolate was subjected to sequencing of the pbp1a gene. Primers Amx ResF and Amx ResIIR were used to amplify the 3′ region of the gene, covering nucleotide positions from 910 bp to the terminal end, based on prior studies implicating this region in resistance development [14, 27] Sequence analysis revealed eleven amino acid substitutions within this region of the pbp1a gene.

Functional evaluation of putative resistance-associated mutation by transformation

To assess the role of specific point mutations (Table 3) in conferring resistance, natural transformation experiments were conducted using the amoxicillin-sensitive H. pylori strain 26695 as the recipient. Overlapping PCR fragments encompassing the mutated regions were generated using Amx ResF/R and Amx ResII F/R primers. Post-transformation, colonies were selected on amoxicillin-containing media (at 0.25 µg/ml). Ten resistant colonies were randomly picked and their resistance profiles confirmed via MIC determination. Sequencing of the pbp1a gene in these transformants revealed that only four mutations—E406A, D535N, S543H, and T556S—were consistently present across all resistant transformants, implicating them as putative resistance-determining mutations, consistent with prior literature [14, 27]. Other mutations initially detected in the wild-type resistant strain—including D479E, N504D, and M515I—were not consistently retained in the transformants. These changes, previously identified in amoxicillin-sensitive isolates from Kolkata [16], were therefore excluded as causal mutations in the development of resistance.

Susceptibility to tetracycline and furazolidone

All H. pylori isolates (n = 88) included in the present study were found to be sensitive to both tetracycline and furazolidone. Among the tetracycline-sensitive strains, a majority (83 out of 88; 94.3%) exhibited a MIC range between 0.125 µg/ml and ≤ 0.5 µg/ml. The remaining 5 strains (5.7%) were also sensitive, with MIC values ranging from 0.5 µg/ml to ≤ 1.0 µg/ml. Similarly, out of the 88 furazolidone-sensitive strains, 71 isolates (80.7%) demonstrated MIC values in the range of 0.0625 µg/ml to ≤ 0.125 µg/ml. The remaining 17 strains (19.3%) had MIC values between 0.125 µg/ml and ≤ 0.25 µg/ml, as illustrated in Fig 1.

Prevalence of multi and dual drug resistant strains

A total of 9 out of 88 isolates (10.2%) were identified as multidrug-resistant (MDR), defined as resistant to at least three classes of antibiotics. Among these, 8 strains exhibited simultaneous resistance to metronidazole, clarithromycin, and levofloxacin, while a single strain showed combined resistance to metronidazole, amoxicillin, and levofloxacin. In addition to the MDR isolates, dual-drug resistance was also observed in several strains. The most common dual resistance pattern involved metronidazole and levofloxacin, identified in 29 out of 88 isolates (33%). Furthermore, dual resistance to clarithromycin and levofloxacin was detected in 6 isolates (6.8%). Only one isolate showed dual resistance to both metronidazole and clarithromycin. A detailed summary of the resistance profiles is provided in Table 2

Comparative analysis of antibiotic resistance profile with cagA and vacA genotypes

As shown in Table 4, the distribution of cagA and vacA genotypes among the 88 H. pylori isolates demonstrated that four genotype combinations were represented across the study population, irrespective of their antibiotic susceptibility profiles. The combinations included cagA-positive and -negative strains along with varying vacA allelic types (s1m1, s1m2, s2m2). This uniform distribution suggests no exclusive association between any specific cagA/vacA genotype and overall antibiotic resistance. However, a noteworthy observation was made within the subset of strains harboring the cagA-positive/vacA s1m1 genotype. Out of the 50 isolates belonging to this group, 20 strains (40%) exhibited dual resistance to both metronidazole and levofloxacin. Despite this, statistical analysis did not indicate a significant association between this genotype and dual-drug resistance, and no specific cagA/vacA combination was consistently linked to resistance against any single antibiotic.

Table 4.

Antibiotic susceptibility pattern w.r.t. resistance category

Strain genotype (n = 88)	Resistance category	Strain number
cagA (+ ve); vacA (s1m1) (n = 50)	MET + CLA + LVX	2 (4%)
	MET + LVX + AMX	1 (2%)
	MET + LVX	20 (40%)
	CLA + LVX	4(8%)
	MET + CLA	1(2%)
	MET	9 (18%)
	CLA	1 (2%)
	LEV	9 (18%)
	SENSITIVE	3 (6%)
cagA (+ ve); vacA (s1m2) (n = 15)	MET + CLA + LVX	3(20%)
	MET + LVX	5 (33%)
	CLA + LVX	1(6.7%)
	MET	3 (20%)
	LEV	2 (13.3%)
	SENSITIVE	1 (%)
cagA (–ve); vacA (s2m2) (n = 19)	MET + CLA + LVX	3 (15.7%)
	MET + LVX	3 (15.7%)
	CLA + LVX	1 (5.3%)
	MET	2 (10.5%)
	CLA	1 (5.3%)
	LVX	7 (36.8%)
	SENSITIVE	2 (10.5%)
cagA (–ve); vacA (s1m1) (n = 3)	MET + LVX	1 (33.3%)
	MET	1 (33.3%)
	SENSITIVE	1 (33.3%)
cagA (–ve); vacA (s1m2) (n = 1)	SENSITIVE	1 (100%)

The distribution of Helicobacter pylori strains (n = 88) based on various antibiotic resistance patterns in relation to the virulence genotype combinations of cagA and vacA (as indicated in parentheses)

Discussion

Antibiotic resistance in H. pylori is a growing concern, driven by misuse of antibiotics in healthcare and agriculture, as well as patient non-compliance. The bacterium’s genetic variability leads to region-specific differences in susceptibility [16]. In many developing countries, empirical treatment without prior susceptibility testing reduces therapeutic efficacy. The COVID-19 pandemic has worsened the situation through excessive community use of macrolides, particularly azithromycin, valued for its anti-inflammatory and antiviral properties [28]. Such use may promote cross-resistance with clarithromycin, potentially increasing clarithromycin resistance [28]. The emergence of drug-resistant H. pylori strains has serious clinical implications, including treatment failures, persistent infections, aggravated gastrointestinal symptoms, and increased healthcare costs. Furthermore, these resistant strains can disseminate within communities, making eradication efforts even more challenging. Therefore, continuous, region-specific surveillance of resistance patterns is crucial for designing evidence-based treatment guidelines and mitigating the growing burden of antibiotic resistance.

High clarithromycin resistance posing great threat

In our study, resistance rate to clarithromycin was observed in 19.3% of the H. pylori isolates, posing a significant threat. This figure not only exceeds the empirical resistance threshold [12] but also marks a stark rise from the 0% resistance reported earlier in Kolkata [17]. A similar upward trend has been documented in other regions such as Korea, where resistance rate increased from 37% in 2012 [29] to a troubling 71.1% by 2019 [30]. Across India, clarithromycin resistance is increasingly prevalent, with reported rates in Karnataka (20.4%), Odisha (34.4%), Tamil Nadu (8.7%), North East India (6.5%), and Delhi (11.8%) [10, 31–34]. Comparable data from neighboring countries reflect this growing resistance: Myanmar (7.7%), Bangladesh (39.3%), Nepal (21.4%) and Pakistan (5.4%) [35–37]. Globally, resistance rates are similarly high: China reports 32.4% [38], Africa, 29.2%, Europe, 34%; and the Americas, 14% resistance [13, 39]. These figures collectively validate the World Health Organization’s classification of H. pylori as a high-priority pathogen.

Our study identified the A2143G mutation in the 23SrRNA gene as the predominant genetic determinant of clarithromycin resistance, found in 15 out of 17 (88%) resistant strains. This finding aligns with previous data from China [40]. This strong correlation between clarithromycin resistance and the A2143G mutation was confirmed using a combination of novel PCR assays (including PCR-RFLP), and Sanger sequencing, all showing 100% concordance. The remaining 2 (12%) resistant strains did not show any positive band in this PCR which implies resistance in these strains may be mediated by alternative mutations in the 23SrRNA gene or through other, as-yet-uncharacterized mechanisms, highlighting the complexity and evolving nature of antibiotic resistance in H. pylori. Other mutations, including A2182C, A1821G, G1826A, and T1830C, were detected in both resistant and sensitive strains, corroborating the previous findings [41–44], suggesting they may not be directly involved in clarithromycin resistance but rather represent naturally occurring polymorphisms within the H. pylori 23SrRNA gene. Thus, controversy lies regarding the roles of these polymorphic forms in clarithromycin resistance development. The almost perfect agreement, quantified by a Cohen’s Kappa analysis value of 1, underscores the reliability of these diagnostic tools. Furthermore, transformation experiments demonstrated that the A2143G mutation alone is sufficient to confer clarithromycin resistance, indicating the potential for rapid and widespread dissemination of resistance in natural populations. Considering the limited availability of sequencing facilities in many parts of India, these alternative molecular diagnostic methods such as restriction enzyme digestion and MAMA-PCR offer feasible, specific, and cost-effective options for the detection of clarithromycin resistance-associated mutations.

A substantial decline in metronidazole resistance, yet above 15% threshold

In contrast to clarithromycin, metronidazole, although highly variable across regions, remains consistently elevated in economically disadvantaged countries [15]. Our current findings indicate a 61.4% metronidazole resistance rate in WB, a substantial decline compared to earlier reports from 2005 [1, 17], likely reflecting reduced clinical use for parasitic and anaerobic infections.

Nonetheless, very high metronidazole resistance has been reported from other parts of India, such as Odisha (81.2%) and Karnataka (81.4%) [31, 32], and from neighbouring countries including Myanmar (80%), Bangladesh (94.6%), Nepal (88.1%), and Pakistan (89%) [35, 36, 45]. High resistance is also reported in China (90.1%) and Korea (67.7%), as well as across Africa (75.8%) [30, 38, 39]. In contrast, metronidazole resistance rates are relatively lower in developed regions such as Europe (38%) and the Americas (27%) [13]. Despite a reduction in resistance among WB isolates, the levels remain well above the 15% threshold, rendering the drug unsuitable for empirical inclusion in eradication regimens.

Consistent with this, our sequencing data revealed that two highly resistant H. pylori strains with an intact frxA harbored mutations in the rdxA, reaffirming its primary role in metronidazole resistance.

Metronidazole resistance poses a major obstacle in the efficacy of standard triple therapy for H. pylori eradication, increasing the risk of treatment failure by approximately 2.5-fold [15]. Furthermore, metronidazole resistance undermines the success of sequential therapy and may even contribute to the emergence of primary clarithromycin resistance, compounding the challenge of effective treatment [19]. However, traditional bismuth-containing quadruple therapies (BQTs), which use higher doses of metronidazole (1500–1600 mg/day), can effectively address this issue [29].

Levofloxacin, one of the most commonly used over-the-counter antibiotics justified the alarmingly high resistance in WB

Second-line regimens combining PPIs, bismuth, amoxicillin, and levofloxacin can achieve up to 90% eradication after triple therapy failure [11]. However, rising levofloxacin resistance threatens their effectiveness. In our study, 69.3% of WB isolates were resistant. Similar increases have been reported in Northeast India, where resistance rose from 73.2% in 2016 to 89.1% in 2019 [34]. High rates are also seen in Karnataka (54.9%) and Odisha (65.6%) [31, 32], while Gujarat reports a lower rate of 13.8% [46]. Neighboring countries, including Bangladesh (66%), Nepal (43%) [47], Myanmar (33.8%) [35], and Pakistan (76%) [45], also face substantial resistance. These findings emphasize the need for routine susceptibility testing and judicious fluoroquinolone use to preserve second-line therapies.

The global surge in fluoroquinolone consumption, an estimated 64% increase between 2000 and 2010, is likely attributable to the widespread use of fluoroquinolone-monotherapy as an alternative first-line treatment for community-acquired pneumonia [47]. In our local population, levofloxacin is among the most commonly used over-the-counter antibiotics, frequently prescribed for upper respiratory tract infections such as coughs and colds, contributing to escalating resistance levels.

Molecular analysis revealed two common GyrA mutations, occurring at amino acid positions 87 and 91. Similar to findings in Japan [22], the mutation at position 91 in WB isolates was associated with low-level resistance, with MIC values ranging from 2 µg/ml to ≤ 4–8 µg/ml. However, additional mutations at position 87 led to significantly higher MICs, ranging from 16 µg/ml to ≤32 µg/ml. Strains with a single mutation at position 87 exhibited MICs as high as 8–32 µg/ml, suggesting that the 87^th amino acid substitution plays a critical role in not only conferring levofloxacin resistance but also elevating the resistance level beyond that observed with the 91^st position mutation.

Low amoxicillin resistance trend in WB

In terms of amoxicillin resistance, only one resistant strain was identified in our study, marking a slight change from 0% resistance reported in 2005 [17]. In contrast, resistance rates were higher in Odisha (3.1%), Tamil Nadu (24%) and Delhi (17.6%) [10, 31, 33, 48]. Other regions such as Lucknow and Chandigarh reported 100% sensitivity [48], while neighboring countries like Bangladesh (3.6%) and Myanmar (4.6%) also showed low resistance [35, 36]. Several countries including those in Europe, as well as the USA, Japan, and Turkey, continue to report high rates of amoxicillin sensitivity [29]. The consistently low amoxicillin resistance in WB underscores its continued efficacy, though regular monitoring remains essential. Sequencing of the pbp1A gene identified mutations consistently present in all 10 naturally transformed resistant colonies, suggesting a potential role in resistance development. Additionally, transformation-based validation supports the functional involvement of the four identified pbp1A point mutations in mediating amoxicillin resistance and helps distinguish true resistance-associated variants from incidental polymorphisms. However, further functional studies are needed to conclusively determine the significance of these mutations.

All isolates from study population, sensitive to furazolidone and tetracycline

Furazolidone is often considered as an alternative treatment in regions with high metronidazole resistance. Encouragingly, our WB isolates exhibited 100% sensitivity to furazolidone. Similar results have been reported in Bangladesh, Nepal [36], Korea [30] and China [38]. However, resistance has been noted in northern India (22.1%) [10] and Gujarat (13.8%) [46]. Despite noting good sensitivity in WB, the commercial unavailability of furazolidone acts as the rate limiting step in overcoming this therapeutic failure. Tetracycline sensitivity was 100% in our WB isolates, aligning with reports from several other Indian states [48, 49] and neighboring countries [35, 36]. Notably, resistance in Gujarat was reported with an unusually high rate of 50% [46]. The improvement in WB is particularly noteworthy, as resistance has decreased from 7.5% in 2005 [17] to 0% in the current study.

Majority MDR strains in WB showed global MDR profiles with resistance towards metronidazole, clarithromycin,  and levofloxacin

MDR H. pylori represents one of the most pressing challenges in eradication therapy, with resistance patterns varying by region, time, and host-related factors. MDR development is influenced by mechanisms such as coccoid transformation, efflux pump activation, and biofilm formation. The most common global MDR profile involves resistance to clarithromycin, metronidazole, and quinolones—mirrored in our findings, with 88.9% of MDR strains exhibiting resistance to this combination [50]. Similar trends were noted in Karnataka, where 86.6% of MDR strains were resistant to at least metronidazole and levofloxacin [32]. In comparison, primary MDR rates remain ≤ 10% in many European countries, but exceed 40% in Peru, highlighting a global disparity [50].

cagA, vacA lacked any direct association with antimicrobial resistance in WB

Virulence factor screening (cagA, vacA) of the 88 isolates revealed no significant association with resistance phenotypes, indicating that the presence of these genes may not directly influence antimicrobial resistance in our population. These findings align with the conclusions of Lee et al., [7], suggesting that epidemiological profiling alone is insufficient for guiding treatment strategies. Instead, region-specific antibiotic susceptibility testing should be a prerequisite for effective eradication therapy. In addition, we identified cases in which multiple H. pylori strains co-infected a single host, each exhibiting distinct antibiotic susceptibility profiles. This phenomenon significantly complicates treatment planning and highlights the importance of comprehensive diagnostics. If not properly monitored and managed with judicious antibiotic use, cases such as this are likely to accelerate disease progression and contribute to the further spread of resistance.

Hence, its high time to buckle up and address this resistance crisis with great care both by formulating therapeutic regimen in correct combination and by ensuring rapid identification of drug resistance with a constant monitoring and sustained surveillance. The global surge in macrolide and fluoroquinolone usage has significantly contributed to resistance trends, underscoring the urgent need for judicious antibiotic stewardship. Alternatives such as bismuth-containing quadruple-therapy must be considered more routinely, particularly in high-resistance regions. Finally, combating the emergence and spread of drug-resistant H. pylori demands a coordinated and multi-sectoral approach integrating healthcare providers, policymakers, researchers, and public health awareness initiatives. In parallel, continued research into the molecular mechanisms and genetic basis of resistance will be critical for developing targeted, next-generation therapies and ensuring sustained success in H. pylori eradication efforts.

While this study provides valuable insights into the antimicrobial resistance (AMR) patterns and genetic mutations in H. pylori strains isolated from patients in WB, Eastern India, several limitations must be acknowledged. The isolates analyzed were obtained from a specific region, and therefore, the findings may not be representative of other regions in India or globally. Regional variations in antibiotic usage and genetic diversity of H. pylori may influence resistance patterns. Resistance mechanisms beyond point mutations, such as efflux pump activity or epigenetic modifications, were not explored and may contribute to the observed resistance. Finally, the study did not integrate patient clinical data, such as treatment history or therapeutic outcomes, which limits the clinical interpretability of these findings.

Conclusions

The alarmingly high rate of clarithromycin resistance (19.3%) among H. pylori isolates from WB is predominantly associated with the A2143G mutation in the 23SrRNA gene. Primary resistance to metronidazole (61.4%) and levofloxacin (69.3%) was also found to be very high, suggesting that these antibiotics may be unsuitable for use in first-line therapy in this region. While resistance to amoxicillin was detected in only a single isolate, all strains exhibited 100% sensitivity to both furazolidone and tetracycline, highlighting their potential as alternative therapeutic options. The correlation between rdxA mutations and metronidazole resistance observed, was remarkably stronger in comparison to frxA mutations. Notably, a novel mutation at the 85^th amino acid position of GyrA was identified in one levofloxacin-resistant strain; however, further studies are needed to confirm its role in resistance development. Screening for virulence-associated genes cagA and vacA did not indicate any significant association with antibiotic resistance in this study population. Collectively, these findings underscore the urgent need to develop region-specific treatment regimens based on local resistance patterns, emphasizing the rational and judicious use of antibiotics in the management of H. pylori infection.

Author contributions

SP, BCK and AKM developed the conceptual framework for the current study and decided on the approaches. Acquisition of laboratory data, was done by BCK, SP and NR while clinical data acquisition was done by SC. SP and BCK carried out the experiments and performed the analysis. SP, BCK, AKM collaboratively created the original draft. BCK, SP, AKM all contributed to the editing and revision of the text. AKM supervised the project. All authors reviewed the manuscript and provided consent for communication to journal.

Funding

This work was supported in part by the Council of Scientific and Industrial Research (CSIR) [File No. 09/482(0065)/2017-EMR-I] and Indian Council of Medical Research (ICMR), Government of India. BCK acknowledges Indian Council of Medical Research (ICMR) for providing a Senior Research Fellowship (No. 5/3/8/36/ITR-F/2020)/Department of Science and Technology for providing fellowship (DST/INSPIRE Fellowship/2014/IF140909). NR acknowledges UGC for providing fund NTA Ref. No.:201610173886. Funders have no role in study design, data collection, analysis, interpretation and publication.

Data availability

The sequences submitted in the GenBank are available with accession numbers PQ062041-PQ062051 for *23SrRNA* gene, PQ083119-PQ083124 for reverse complementary sequences of *rdxA,* and PQ083112-PQ083117 for *frxA,* PQ083084-PQ083110 for *gyrA* gene, PQ083118 for *pbp1a* gene. The original data and any other clarification for this work are available upon email request to the corresponding author.

Declarations

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the ICMR-National Institute of Cholera and Enteric Diseases (ICMR-NICED) Ethical committee, Manipal Hospitals (formerly AMRI Hospital) committee [Ref: AMRI -EC/AP-49/2019-20] and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

Written informed consent for publication of their clinical details, that their samples were obtained solely for research purpose, was obtained from the patient/parent/guardian/relative of the patient. A copy of the consent form is available for review by the Editor of this journal.

Competing interests

Asish Kumar Mukhopadhyay is an Associate Editor for Gut Pathogens and was not involved in the peer review of this paper. The authors declare that they have no other competing interests.